Anti-thrombotic and vascular effects of AR246686, a novel 5-HT 2A receptor antagonist John W. Adams ⁎ , Juan Ramirez, Danny Ortuno, Yunqing Shi, William Thomsen, Jeremy G. Richman, Michael Morgan, Peter Dosa, Bradley R. Teegarden, Hussien Al-Shamma, Dominic P. Behan, Daniel T. Connolly Arena Pharmaceuticals, Inc. San Diego, CA 92121 USA Received 30 July 2007; received in revised form 13 November 2007; accepted 21 November 2007 Available online 3 December 2007 Abstract We have evaluated the anti-platelet and vascular pharmacology of AR246686, a novel 5-hydroxytryptamine2A (5-HT 2A ) receptor antagonist. AR246686 displayed high affinity binding to membranes of HEK cells stably expressing recombinant human and rat 5-HT 2A receptors (K i = 0.2 nM and 0.4 nM, respectively). Functional antagonism (IC 50 = 1.9 nM) with AR246686 was determined by inhibition of ligand- independent inositol phosphate accumulation in the 5-HT 2A stable cell line. We observed 8.7-fold and 1360-fold higher affinity of AR246686 for the 5-HT 2A receptor vs. 5-HT 2C and 5-HT 2B receptors, respectively. AR246686 inhibited 5-HT-induced amplification of ADP-stimulated human platelet aggregation (IC 50 = 21 nM). Similar potency was observed for inhibition of 5-HT stimulated DNA synthesis in rat aortic smooth muscle cells (IC 50 =10 nM) and 5-HT-mediated contraction in rat aortic rings. Effects of AR246686 on arterial thrombosis and bleeding time were studied in a rat model of femoral artery occlusion. Oral dosing of AR246686 to rats resulted in prolongation of time to occlusion at 1 mg/kg, whereas increased bleeding time was observed at a dose of 20 mg/kg. In contrast, both bleeding time and time to occlusion were increased at the same dose (10 mg/kg) of clopidogrel. These results demonstrate that AR246686 is a high affinity 5-HT 2A receptor antagonist with potent activity on platelets and vascular smooth muscle. Further, oral administration results in anti-thrombotic effects at doses that are free of significant effects on traumatic bleeding time. © 2007 Elsevier B.V. All rights reserved. Keywords: 5-hydroxytryptamine; 5-HT 2A receptor; Platelet; Thrombosis; Vasoconstriction; Serotonin; Platelet aggregation; Bleeding time 1. Introduction Serotonin (5-hydroxytryptamine, 5-HT) is a naturally occurring indoleamine found primarily in the brain, entero- chromaffin tissue and platelets. 5-HT exerts a multitude of biological effects achieved through interaction with specific cell surface G-protein coupled receptors. To date, at least 14 different human 5-HT receptors are known (Kaumann and Levy, 2006). Amongst them, 5-HT 2A receptors on vascular smooth muscle cells and platelets play an important role in regulation of cardiovascular function. The uptake process and storage capacity of 5-HT in platelets is such that minimal amounts of the amine exists in normal plasma. However, upon platelet activation at the site of vessel injury, 5-HT is released from the dense granules in platelets (Ashton et al., 1986). 5-HT by itself is a weak activator of platelet aggregation but amplifies aggregation induced by other agonists including collagen, ADP, epinephrine and thrombin (De Clerck and Herman, 1983; De Clerck and Janssen, 1990). Thus, subsequent to platelet adhesion and activation at the site of vascular injury, 5-HT released from platelets induces further platelet aggregation and enhances thrombogenesis. This phenomenon is supported by clinical data demonstrating that increased blood serotonin levels correlate with cardiac events (Vikenes et al., 1999). Moreover, hyperactive 5-HT 2A receptor activity has been implicated in the increased coronary events Available online at www.sciencedirect.com European Journal of Pharmacology 586 (2008) 234 – 243 www.elsevier.com/locate/ejphar ⁎ Corresponding author. Cardiovascular Biology, Arena Pharmaceuticals, Inc. San Diego, CA 92121 USA. Tel.: +1 858 453 7200. E-mail address: jadams@arenapharm.com (J.W. Adams). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.11.056